Antioxidant activity and free radical-scavenging capacity ofextracts from guava (Psidium guajava L.) leaves

Hui-Yin Chen a,*, Gow-Chin Yen b

a Department of Food Science, Central Taiwan University of Science and Technology, 11, Putzu Lane, Peitun District, Taichung 40601, Taiwan, ROCb Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung, Taiwan, ROC

Received 8 November 2004; received in revised form 7 February 2006; accepted 7 February 2006

Abstract

The objectives of this study were to study the antioxidant activity and free radical-scavenging effects of extracts from guava leaves anddried fruit. The results indicated that 94.4–96.2% of linoleic acid oxidation was inhibited by the addition of guava leaf and guava teaextracts at a concentration of 100 lg/ml. The guava dried fruit extracts exhibited weaker antioxidant effects than did the leaf extracts.The results also demonstrated that the scavenging effects of guava leaf extracts on ABTS�+ radicals and superoxide anion increased withincreasing concentrations. The guava leaf extracts displayed a significant scavenging ability on the peroxyl radicals. However, the scav-enging effects were decreased when the extract concentration was greater than 10 lg/ml. The extracts from leaves of various guava cul-tivars exhibited more scavenging effects on free radicals than did commercial guava tea extracts and dried fruit extracts. Thechromatogram data indicated that guava extracts contained phenolic acids, such as ferulic acid, which appeared to be responsible fortheir antioxidant activity. Correlation analysis indicated that there was a linear relationship between antioxidant potency, free radi-cal-scavenging ability and the content of phenolic compounds of guava leaf extracts.� 2006 Published by Elsevier Ltd.

Keywords: Guava leaves; Antioxidant activity; Radical scavenging; Phenolic compound

1. Introduction

The involvement of active oxygen and free radicals inthe pathogenesis of certain human diseases, including can-cer, aging and atherosclerosis is increasingly being recog-nized (Moskovitz, Yim, & Chock, 2002). Active oxygenand free radicals, such as superoxide anion ðO��2 Þ, hydro-gen peroxide (H2O2) and hydroxyl radical (�OH), are con-stantly formed in the human body by normal metabolicaction. Their action is opposed by a balanced system ofantioxidant defences, including antioxidant compoundsand enzymes. Upsetting this balance causes oxidativestress, which can lead to cell injury and death (Halliwell& Gutteridge, 1999). Therefore, much attention has been

0308-8146/$ - see front matter � 2006 Published by Elsevier Ltd.

doi:10.1016/j.foodchem.2006.02.047

* Corresponding author. Tel.: +886 4 22391647 7501 19; fax: +886 422396771.

E-mail address: hychen2@ctust.edu.tw (H.-Y. Chen).

focussed on the use of antioxidants, especially natural anti-oxidants, to inhibit lipid peroxidation, or to protect againstthe damage of free radicals (Vendemiale, Grattagliano, &Altomare, 1999). Current research into free radicals hasconfirmed that foods rich in antioxidants play an essentialrole in the prevention of cardiovascular diseases and can-cers (Kris-Etherton et al., 2002) and neurodegenerative dis-eases (Di Matteo & Esposito, 2003).

Recently, there is strong evidence to show that diabetesis associated with increased oxidative stress (Abou-Seif &Youssef, 2004; Freitas, Filipe, & Rodrigo, 1997). More-over, the generation of oxidative stress may play an impor-tant role in the etiology of diabetic complications, such asvascular complications (Cooper, Bonnet, Oldfield, &Jandeleit-Dahm, 2001), diabetic cataract (Ozmen, Mutaf,Ozmen, Mentes, & Bayindir, 1997) and diabetic nephropa-thy (Ha & Kim, 1999). Administration of streptozotocininduces diabetes in experimental animals. The development

of diabetes induced by streptozotocin was related to theproduction of radicals, including superoxide anion andhydroxyl radicals (Chang et al., 1993; Ohkuwa, Sato, &Naoi, 1995). Kakkar, Mantha, Radhi, Prasad, and Kalra(1997) also reported that the lipid peroxide level in the kid-ney of streptozotocin-induced diabetic rats was signifi-cantly higher than that of the control (P < 0.05).Evidence indicates that many biochemical pathways associ-ated with hyperglycemia can increase the production offree radicals (Lee & Chung, 1999). Diabetic patients havereduced antioxidant defences and suffer from an increasedrisk of free radical-mediated damage (Maxwell et al., 1997).The levels of plasma lipid peroxide products, includingmalondialdehyde in diabetic patients, were increased whencompared with control subjects (Freitas et al., 1997). For-tunately, supplementation of vitamin E can lower lipid per-oxidation in diabetic patients (Jain et al., 1996). Noda,Mori, and Packer (1997) indicated that gliclazide, com-monly used in the treatment of diabetes, was not only effec-tive in reducing blood sugar, but also might be useful in thescavenging of free radicals. Armstrong, Chestnutt, Gorm-ley, and Young (1996) suggested that an improved antiox-idant status and reduced lipid peroxidation might be onemechanism by which dietary treatment contributes to theprevention of diabetic complications.

Recently, research on phytochemicals and their effectson human health have been intensively studied. In particu-lar, research has focussed on a search for antioxidants,hypoglycemic agents, and anticancer agents from vegeta-bles, fruit, tea, spices and medicinal herbs. Prince andMenon (1999) showed that oral administration of aqueousTinospora cordifolia root extract, an indigenous plant usedas medicine in India, resulted in a decreased level ofTBARS and an increase in the levels of glutathione andvitamin C in alloxan diabetes. The extracts from neem(Azadirachta indica A. Juss Meliaceae) seed also expressedsignificant protection against the oxidative stress damageinduced by streptozotocin in the heart and erythrocytesof rats (Gupta, Kataria, Gupta, Murganandan, & Yash-roy, 2004). Guava (Psidium guajava L.) is widely cultivatedand its fruit is popular in Taiwan. Guava was also used as ahypoglycemic agent in folk medicine. The leaves and skinof the fruit have greater effects. Guava tea, the infusionof dried guava fruit and leaves, has recently become popu-lar as a drink in Taiwan. Cheng and Yang (1983) provedthat guava juice exhibited hypoglycemic effects in mice.Interestingly, the decreased serum glucose level of infusionsfrom the African mistletoe (Loranthus bengwensis L.) para-site on guava trees was more affected than that preparedfrom mistletoe parasitic on other trees (Obatomi, Bikomo,& Temple, 1994). In other studies, the anti-diarrhoeal(Lutterodt, 1989) antipyretic (Olajide, Awe, & Makinde,1999), antimicrobial (Jaiarj et al., 1999) and bio-antimuta-genic (Matsuo, Hanamure, Shimoi, Nakamura, & Tomita,1994) properties of guava leaf extract have been demon-strated. There is an important role of oxidative stress inthe development of cancer and diabetes. As noted above,

the infusion of guava leaves has potential as a functionaldrink. However, information concerning the antioxidantactivity of guava leaves is unavailable. The objectives ofthis study were to study the antioxidant activity and freeradical-scavenging effects of guava leaf and dried fruitextracts.

2. Materials and methods

2.1. Chemicals

2,2 0-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS), ferrozine, linoleic acid, nitro blue tetrazolium(NBT), peroxidase, b-phycoerythrin and polyphenon 60were purchased from Sigma Chemical Co. (St. Louis,MO, USA). 2,2-Azobis(2-amidinopropane) dihydrochlo-ride (AAPH) was purchased from Wako Pure ChemicalCo. (Tokyo, Japan). Ammonium thiocyanate, dihydro-nic-otin-amidadenin-dinucleotide (NADH), Folin–Ciocalteaureagent and phenazine methosulphate (PMS) were pur-chased from E. Merck Co. (Darmstadt, Germany).

2.2. Plant material and extraction

Fresh leaves of various guava cultivars, including HongBa, Shi Ji Ba, Shui Jing Ba and Tu Ba, were provided byFengshan Tropical Horticultural Experiment Branch ofthe Agricultural Research Institute, Taiwan. The commer-cial products, guava tea A (dried leaves), guava tea B (driedleaves:dried fruit = 1:1; w/w) and dried fruit were pur-chased at a local market in Taichung, Taiwan. The freshguava leaves were dried at 40 �C for 12 h in an oven andthen broken into pieces of about 0.5 cm2. Samples (20 g)were extracted with boiling water (500 ml) for 5 min. Theextracts were filtered through Whatman No. 2 filter paperand the filtrates were freeze-dried.

2.3. Antioxidant activity in a linoleic acid system

The antioxidant activities of extracts from guava weredetermined by the thiocyanate method (Mitsuda, Yasum-oto, & Iwami, 1966). Each sample, in 0.5 ml of distilledwater, was mixed with linoleic acid emulsion (2.5 ml0.02 M, pH 7.0) and phosphate buffer (2 ml, 0.2 M, pH7.0) in a test tube and stood, in darkness, at 37 �C, toaccelerate oxidation. The linoleic acid emulsion was pre-pared by mixing an equivalent weight of linoleic acidand Tween 20 in phosphate buffer (0.2 M, pH 7.0). Theperoxide value was determined by reading the absorptionat 500 nm with a spectrophotometer (Hitachi U-2000)after colour development with FeCl2 and thiocyanate atvarious intervals during incubation. The peroxidation oflinoleic acid was calculated as peroxidation (%) = (A1/A0) · 100, where A0 was the absorption of the controlreaction and A1 was the absorption in the presence ofsample. All analyses were run in triplicate and mean val-ues were calculated.

2.4. Radical-scavenging assays

2.4.1. Scavenging effects on ABTS�+ radicals

This decoloration method consists of an enzymatic sys-tem containing a peroxidase, hydrogen peroxide andABTS. A radical is generated from ABTS and has a char-acteristic absorption spectrum with a maximum of414 nm. The ability of extracts from guava to scavengeABTS�+ radicals was determined by the method describedin the work of Okamoto, Hayase, and Kato (1992) withsome modifications. In brief, 30 lM H2O2 and extractswere added to 0.1 M phosphate buffer (pH 6.0) mediumcontaining 0.02% ABTS and 6 units of peroxidase. Absor-bance at 414 nm was measured after incubation for15 min. All analyses were run in triplicate and mean val-ues were calculated.

2.4.2. Scavenging effects on superoxide anion

The influence of extracts from guava on the generationof superoxide anion was measured according to the methoddescribed in previously work (Yen & Chen, 1995). Super-oxide anion was generated in a non-enzymic system anddetermined by a spectrophotometric measurement forreduction of nitro blue tetrazolium. The reaction mixture,which contained 1 ml of extract in distilled water, 1 ml ofPMS (60 lM) in phosphate buffer (0.1 M, pH 7.4), 1 mlof NADH (468 lM) in phosphite buffer and 1 ml of NBT(150 lM) in phosphate buffer, was incubated at ambienttemperature for 5 min, and the colour was read at560 nm against blank samples. All analyses were run intriplicate and mean values were calculated.

2.4.3. Scavenging effects on peroxyl radicals

The ability of extracts from guava to scavenge peroxylradicals was measured by monitoring the loss of b-phyco-erythrin fluorescence induced by AAPH. The Fluorescentdegradation of b-phycoerythrin was measured at 540 nmexcitation and 575 nm emission (Glazer, 1990). The reac-tion mixture contained 5 · 10�10 M b-phycoerythrin,25 mM AAPH and extracts in 75 mM phosphate bufferat pH 7.4. b-Phycoerythrin fluorescence was almostlost by incubation with 25 mM AAPH for 30 min. Allanalyses were run in triplicate and mean values werecalculated.

2.5. Determination of total phenolic compounds

Total phenolic compounds in the extracts from guavawere determined with Folin–Ciocalteu reagent using gallicacid and (+)-catechin as the standards (Taga, Miller, &Pratt, 1984). Extracts (100 ll) were added to 2 ml of 2%Na2CO3. After 2 min, 50% Folin–Ciocalteau reagent(100 ll) was added to the mixture which was then left tostand for 30 min. Absorbance was measured at 750 nmon a spectrophotometer and compared to gallic acid and(+)-catechin calibration curves. All analyses were run intriplicate and mean values were calculated.

2.6. HPLC analysis of phenolic compounds

The contents of phenolic compounds in extracts fromguava were determined by HPLC, performed with a Hit-achi liquid chromatograph (Hitachi, Ltd., Tokyo, Japan)consisting of a model L-6200 pump, and a model L-4200UV–Vis detector set at 285 nm. The analyses were carriedout on a LiChrospher RP-18 column (250 mm · 4 mmi.d., 5 lm, E. Merck Co., Darmstadt, Germany). Extractswere filtered through a 0.45 lm filter before use. The elu-tion solvents were (A) 0.2 M NaH2PO4 adjusted to pH3.0 by phosphoric acid, and then diluted with distilledwater to 50 mM NaH2PO4 and (B) 50 mM NaH2PO4/methanol/acetonitrile (30/20/50, v/v/v). The solvent gradi-ent elution programme used was as follows: initial 100% A,hold for 6 min; linear gradient to 92% A in 8 min and holdfor 6 min; linear gradient to 82% A in 35 min; linear gradi-ent to 62% A in 10 min and hold for 15 min; linear gradientto 0% A in 10 min and hold for 10 min. The flow rate was1 ml/min. Phenolic compounds were identified by compar-ison of their retention time (Rt) values and UV spectra withthose of known standards and determined by peak areasfrom the chromatograms. All analyses were run in tripli-cate and mean values were calculated.

2.7. Statistical analysis

Data were analyzed using the Statistical Analysis Systemsoftware package. Analyses of variance were performedusing ANOVA procedures. Significant differences betweenmeans were determined using Duncan’s multiple range test.

3. Results and discussion

3.1. Antioxidant activity in a linoleic acid system

The antioxidant activities of extracts from guava, deter-mined using the thiocyanate method, were compared withthat of polyphenon 60, which is a commercial polyphenolproduct extracted from green tea. Comparison of antioxi-dant activity of extracts from guava at various concentra-tions is shown in Table 1. In all samples, the extractsfrom Shi Ji Ba showed a stronger antioxidant activity thandid other samples at 50 lg/ml. However, the extracts fromguava leaves and dried fruit all exhibited over 95% inhibi-tion at a concentration of 200 lg/ml. Except for the driedfruit extracts, the other samples were found to have weakerantioxidant activity when the concentration was over200 lg/ml. The polyphenon 60 showed more significantlyprooxidant effects and the maximum antioxidant activitywas weaker than those of extracts from guava leaves andguava tea at a concentration of 100 lg/ml. The extractsfrom dried fruit of guava displayed weaker antioxidantactivity than did other samples at 50 lg/ml. However, therewas no significant difference in antioxidant activity(P > 0.05) between dried fruit of guava and other guavaextracts at a concentration of 200 lg/ml.

Table 1Antioxidant activities of extracts from leaves and dried fruit of guava

Sample concentration (lg/ml) Peroxidation (%)a

Shi Ji Ba Shui Jing Ba Tu Ba Hong Ba Guava tea A Guava tea B Dried fruit Tea polyphenon 60

50 9.3 ± 0.4 14.3 ± 1.9 13.0 ± 2.6 15.6 ± 1.1 20.6 ± 2.9 17.8 ± 2.6 49.9 ± 2.7 17.8 ± 1.8100 3.8 ± 0.1 4.0 ± 0.2 4.4 ± 0.4 5.0 ± 0.3 5.7 ± 1.3 4.7 ± 0.2 11.6 ± 0.8 10.1 ± 1.1150 4.5 ± 0.1 4.1 ± 0.1 4.6 ± 0.1 4.9 ± 0.2 3.8 ± 0.1 3.7 ± 0.2 7.3 ± 0.3 11.3 ± 0.4200 4.7 ± 0.2 4.8 ± 0.4 4.8 ± 0.1 4.5 ± 0.1 3.4 ± 0.2 4.5 ± 0.1 4.8 ± 0.2 13.1 ± 0.4500 9.0 ± 0.3 8.0 ± 0.1 8.7 ± 0.3 8.9 ± 0.1 6.3 ± 0.2 6.2 ± 0.2 0.7 ± 0.1 19.5 ± 1.2

a Peroxidation (%) = [(absorbance of sample at 500 nm)/(absorbance of control at 500 nm)] · 100. A low peroxidation (%) indicated a high antioxidantactivity.

Recently, there has been considerable interest in preven-tive medicine through the quest for natural antioxidantsfrom plant material. Various phytochemical components,such as flavonoids, phenolic acids and carotenoids, areknown to be responsible for the antioxidant capacity ofplants. However, the effectiveness of flavonoids as effectiveantioxidants is dependent upon the environment. A num-ber of factors may influence the behaviour of flavonoidsand may result in alterations to their efficacy as antioxi-dants. The antioxidant activity of flavonoids may bereduced by the autoxidation of flavonoids, catalyzed bytransition metals to produce superoxide anion. The latterdismutates to generate hydrogen peroxide and form hydro-xyl radicals via a Fenton reaction in the presence of transi-tion metals (Canada, Giannella, Nguyen, & Mason, 1990).Most plant polyphenol compounds possess both antioxi-dant and prooxidant properties, depending on concentra-tion and environmental factors (Cao, Sofic, & Prior,1997). A possible mechanism of polyphenol cytotoxicitymay be related to their prooxidant properties. In our previ-ous work, tea extracts showed both antioxidant and proox-idant activities in oxidative damage of biomolecules (Yen,Chen, & Peng, 1997). Azam, Hadi, Khan, and Hadi(2004) proposed that the prooxidant action of tea poly-phenolics may be an important mechanism of their anti-cancer and apoptosis properties. In addition topolyphenolics, the prooxidant activity of green tea extractsmay be caused by their chlorophyll components (Wanas-undara & Shahidi, 1998). In the above results, the extractsfrom guava leaf and fruit exhibited strong potential antiox-idant activity. The true prooxidant effects that guavaextract has on cells remains as a matter to be studiedfurther.

3.2. Radical-scavenging effects

In this study, three free radicals were used to assess thepotential free radical-scavenging activities of guavaextracts, namely ABTS�+ radical, superoxide anion andperoxy radicals. ABTS is a peroxidase substrate which,when oxidized in the presence of H2O2 in a typical peroxi-dative reaction, generates a metastable radical with acharacteristic absorption spectrum and an absorptionmaximum of 414 nm (Arnao, Cano, Hernandez-Ruiz, Gar-cia-Canovas, & Acosta, 1996). The ABTS�+ radicals are

scavenged by antioxidants via the mechanism of elec-tron-/hydrogen-donation and are assessed by measuringthe decrease in absorption at 414 nm. In the superoxideanion-scavenging test, superoxide anion, that is derivedfrom dissolved oxygen through the PMS/NADH couplingreaction, reduces NBT and increases absorption at 560 nm.The decrease in absorption at 560 nm with antioxidantsthus indicates the consumption of superoxide anion. Inthe peroxyl radical-scavenging assay, thermal decomposi-tion of AAPH leads to the formation of carbon-centredradicals which, under aerobic conditions, yield alkylper-oxyl radicals. These radical species can be detected by assayof fluorescent decomposition of b-phycoerthrin, a majorpigment protein of sea algae. The absorption assay forantioxidants was based on oxidation of b-phycoerthrinmolecules by peroxyl radicals (Cao, Alessio, & Cutler,1993).

The abilities of extracts from guava, assayed to be scav-enging the ABTS�+ radical in comparison with polyphenon60, are shown in Fig. 1. The scavenging effect of polyphe-non 60 was observed to be higher than that of extracts fromguava. The polyphenon 60 showed a linear increase inABTS�+ radical-scavenging activity with increasing concen-tration, reaching 97.1 ± 0.9% scavenging activity at a con-centration of 5 lg/ml. In various samples of guava extracts,the scavenging activities of Shi Ji Ba, Hong Ba, and Tu Baextracts on ABTS�+ radicals were stronger than those ofShui Jing Ba, guava tea A and guava tea B extracts. How-ever, the extracts from four guava cultivars expressed over95% scavenging activity at a concentration of 20 lg/ml. Inall samples, extracts from dried guava fruit had the weakestscavenging ability on ABTS�+ radicals. The decolorizationassay, using free blue-green ABTS�+ radicals, was shown tobe a very useful tool in expeditiously measuring the antiox-idant activity of individual chemical compounds or com-plex extracts. This method can express total antioxidantactivity as vitamin C equivalent antioxidant capacity(VCEAC) or as trolox equivalent antioxidant capacity(TEAC) value (Kim, Lee, Lee, & Lee, 2002; Re et al.,1999).

As shown in Fig. 2, the scavenging effects of polyphenon60 and extracts from guava on the superoxide anion weresimilar to the results of the scavenging effects on ABTS�+

radicals. The abilities of all samples to scavenge superoxideanion decreased in the order: polyphenon 60 > Shi Ji Ba,

Concentration (μg /ml)

0 5 10 15 20

Scav

engi

ng e

ffec

ts (

%)

0

20

40

60

80

100

Shi Ji BaShui Jing BaTu BaHong BaGuava tea A Guava tea BDried fruitTea polyphenon 60

Fig. 1. Scavenging effects of extracts from leaves and dried fruit of guava on ABTS�+ radicals.

Concentration (μg /ml)0 100 200 300 400 500

Scav

engi

ng e

ffec

ts (

%)

0

20

40

60

80

100

Shi Ji BaShui Jing BaTu BaHong BaGuava tea AGuava tea BDried fruitTea polyphenon 60

Fig. 2. Scavenging effects of extracts from leaves and dried fruit of guava on superoxide anion.

Time (min)0 2 4 6 8 10

Rel

ativ

e fl

uore

scen

ce (

%)

0

20

40

60

80

100

β -phycoerythrin

β -phycoerythrin + AAPH

C

BD

A

E

Fig. 3. The effects of extracts from leaves of Shi Ji Ba on b-phycoerythrinfluorescence decay induced by AAPH. The concentrations of extracts were(A) 2.5 lg/ml; (B) 5 lg/ml; (C) 10 lg/ml; (D) 25 lg/ml; and (E) 50 lg/ml.

Hong Ba and Tu Ba > Shui Jing Ba, guava tea A andguava tea B > dried fruit.

The influence of Shi Ji Ba extracts on the decrease in thefluorescence of b-phycoerthrin induced by AAPH is shownin Fig. 3. When compared to a time of 0 min, the relativefluorescence of b-phycoerthrin had decreased to 87.7%after incubation for 10 min, indicating that auto-composi-tion of b-phycoerthrin had occurred in the solution. Therapid decomposition of b-phycoerthrin was induced byaddition of AAPH to the solution, and a resulting 2.4%fluorescence remained after incubation for 10 min, whencompared with the control. The extracts from Shi Ji Baexhibited a concentration-dependent biphasic effect onthe fluorescent decomposition of b-phycoerthrin inducedby AAPH. The inhibitions of guava leaf extracts on thefluorescent decomposition of b-phycoerthrin, induced byAAPH, increased with concentration (2.5–10 lg/ml) upto a maximum, and then decreased at the concentration

25–50 lg/ml. This finding was similar to the results of theantioxidant activity assay in a linoleic acid system. Theprooxidant effects of guava extracts at high concentrationsmay be correlated with phenoxy radical formed by thechange of phenolic compounds with phenoxy radicalreacted with b-phycoerythrin to participate in radical chainpropagation. Bowry, Ingold, and Stocker (1992) also indi-cated that a-tocopherol could form a-tocopherol radicaland be a strong prooxidant for low-density lipoprotein athigh concentration of a-tocopherol and low fluxes ofAAPH. The most interesting point from the results(Fig. 3) was the decomposition rate of b-phycoerythrininduced by AAPH in the presence of various concentra-tions of guava leaf extracts. When b-phycoerythrin andAAPH was incubated with guava leaf extracts at 2.5–50 lg/ml (Fig. 3, curves A–E), the fluorescent decomposi-tion of b-phycoerythrin decreased more rapidly than thatof control (b-phycoerythrin + AAPH) after incubationfor 1–2 min. However, with the exception of 5 lg/ml guavaleaf extracts (Fig. 3, curve B), the fluorescent decomposi-tion of b-phycoerythrin, induced by AAPH in the presenceof other concentrations of guava leaf extracts, tended tomoderate at an incubation of 2–10 min. In contrast toother concentrations, curve B showed a linear decreasewith an increase of the incubation time. The reaction ratewas made possible in a different way and resulted a changein expression of the antioxidant activity of samples.

Table 2 summarizes that extracts from guava that inhib-ited the fluorescent decomposition of b-phycoerythrin byAAPH-derived peroxyl radical. With the exception ofpolyphenon 60 and dried fruit, the extracts from four cul-tivars of guava leaves and two kinds of guava tea allshowed over 85% scavenging ability on peroxyl radicalsat a concentration of 10 lg/ml. The extracts from Shi JiBa, Hong Ba and Tu Ba expressed a stronger inhibitionthan that of Shui Jing Ba, guava tea A and guava tea Bat a concentration of 2.5 lg/ml. However, there was no sig-nificant difference in inhibition (P > 0.05) between those sixextracts at a concentration of 10 lg/ml. The inhibitoryeffect of dried fruit extracts was increased with increaseof the concentration. On the contrary, 90.6% fluorescentdecomposition of b-phycoerythrin was inhibited by2.5 lg/ml polyphenon 60, and then decreased with increas-ing concentration. The prooxidant effects of guava leafextracts and polyphenon 60 extracted from green tea leaves

Table 2Scavenging effects of extracts from leaves and dried fruit of guava on the pero

Sample concentration (lg/ml) Scavenging effects (%)a

Shi Ji Ba Shui Jing Ba Tu Ba Hon

2.5 83.3 ± 6.0 77.3 ± 3.1 82.0 ± 5.4 86.15.0 87.6 ± 8.5 84.9 ± 4.5 85.2 ± 2.2 87.7

10.0 88.6 ± 7.9 88.6 ± 4.4 92.5 ± 1.2 94.225.0 74.2 ± 2.4 51.8 ± 3.7 68.1 ± 2.8 77.550.0 58.7 ± 6.9 23.5 ± 2.9 48.5 ± 4.2 58.9

a Scavenging effects (%) = [(fluorescence of b-phycoerythrin with AAPH andphycoerythrin fluorescence of b-phycoerythrin with AAPH)] · 100.

may be attributed to the plant phenolic compounds. How-ever, the components of guava fruit which provided inhib-itory effects on peroxidation remain a matter to be studiedfurther.

As shown above, polyphenon 60 and guava leaf extractsexhibited a concentration-dependent increase in their scav-enging effects on ABTS�+ radicals and superoxide anion.However, the antioxidant activity and radical-scavengingability decreased at high concentrations in the linoleic acidperoxidation and peroxyl radical systems. Comparisonbetween the four methods, ABTS�+ radicals and superoxideanion-scavenging assays was obtained by the absorptionchange induced by the formation of radicals. Nevertheless,antioxidant activity and peroxyl radicals are determined bythe oxidation damage of biomolecular matrices, such aslinoleic acid and protein. The biomolecular matrices maybe attacked by derivatives from sample components, espe-cially the phenolic compounds, resulting in secondary oxi-dation damage. Hanasaki, Ogawa, and Fukui (1994)reported that multiple hydroxyflavonoids, especially withOH in the B-ring, significantly increased production ofhydroxyl radicals in a Fenton system. Liu, Han, Lin, andLuo (2002) demonstrated that 4-hydroxyquinoline deriva-tives could inhibit the free radical-induced peroxidation,but also play a prooxidative role in the vesicle of dipalmi-toyl phosphatidylcholine. This could be due to the elec-tron-attracting group at the ortho position to hydroxylgroup in the phenoxy radical of quinoline derivatives. Athigh concentrations, the phenoxy radical initiated addi-tional propagation of lipid peroxidation. The polyphenon60 and guava leaf extracts showed weaker effects, at highconcentrations, in antioxidant activity and peroxyl radicalscavenging assays. Therefore, the methods and concentra-tions used to measure the antioxidant activity of naturalmaterial, especially polyphenol-rich samples, should takeinto consideration the influence of prooxidant effects.

3.3. Determination of phenolic compounds

Phenolic compounds, such as quercetin, rutin, narigin,catechins, caffeic acid, gallic acid and chlorogenic acid,are very important plant constituents because of their anti-oxidant activities (Croft, 1998; Paganga, Miller, & Rice-Evans, 1999). The analysis of phenolic compounds in theguava extracts is shown in Table 3 and Fig. 4. As shown

xyl radicals

g Ba Guava tea A Guava tea B Dried fruit Tea polyphenon 60

± 4.1 60.2 ± 5.1 37.9 ± 2.4 16.6 ± 7.3 90.6 ± 6.7± 3.6 86.0 ± 6.5 66.8 ± 1.7 44.0 ± 4.3 89.2 ± 4.0± 4.0 90.2 ± 2.5 90.5 ± 3.3 79.3 ± 6.6 79.0 ± 4.6± 5.7 82.2 ± 8.2 89.0 ± 4.9 89.2 ± 2.3 62.9 ± 5.7± 8.7 64.9 ± 7.1 78.7 ± 8.0 91.2 ± 4.2 39.8 ± 5.7

sample-fluorescence of b-phycoerythrin with AAPH)/(fluorescence of b-

Table 3The contents of total phenolic compounds and phenolic acids of extracts from leaves and dried fruit of guava

Samples Total phenolic compounds Phenolic acid

Expressed as (+)-catechin (mg/g) Expressed as gallic acid (mg/g) Gallic acid (lg/g) Ferulic acid (lg/g)

Shi Ji Ba 296 ± 5.4 458 ± 8.1 1621 ± 87.4 672 ± 65.2Shui Jing Ba 267 ± 5.4 414 ± 8.2 793 ± 52.3 108 ± 15.7Tu Ba 313 ± 4.7 483 ± 7.1 1022 ± 62.4 355 ± 45.4Hong Ba 294 ± 4.0 455 ± 6.1 1137 ± 79.6 296 ± 25.9Guava tea A 103 ± 9.3 166 ± 14.1 725 ± 54.1 147 ± 16.8Guava tea B 177 ± 9.2 279 ± 14.0 1278 ± 92.7 234 ± 27.5Dried fruit 69.6 ± 2.8 115 ± 4.2 266 ± 15.4 –Tea polyphenon 60 643 ± 8.5 985 ± 12.8 – –

Fig. 4. HPLC chromatographs of (A) phenolic compound standards and(B) water extracts from Shyj Jih Pa leaves: (1) gallic acid; (2) 3,4-dihydroxybenzoic acid; (3) 4-hydroxybenzoic acid; (4) chlorogenic acid;(5) (+)-catechin; (6) caffeic acid; (7) syringic acid; (8) 2-hydroxybenzoicacid; (9) epicatechin; (10) ferulic acid; (11) naringin; (12) morin; (13)quercetin.

in Table 3, the contents of total phenolic compounds,shown as (+)-catechin equivalents, were less than thoseas gallic acid equivalents. This may be affected by themolecular weight of standards. The contents of total phe-nolic compounds in the extracts were in the order: teapolyphenon 60 > four guava cultivars > guava tea > driedfruit. Tea polyphenon 60, which is a commercial polyphe-nol product extracted from green tea, contained about60% polyphenolic compounds, such as catechins. However,the result from the chromatogram indicated that guavaextracts contained phenolic acids. Fig. 4 shows the chro-matogram of mixed standards (A) and Shi Ji Ba extracts

(B). A good resolution, with sharp peaks, was achievedfor all phenolic compound standards within 90 min. Theresults of HPLC analyses show that three main peaks werefound in the Shi Ji Ba extracts at the absorbance of 285 nm.Gallic acid (Rt = 18.60 min) and ferulic acid (Rt =68.41 min) were identified by comparison of their retentiontime values and UV spectra with those of known standards.The contents of two phenolic acids in guava extracts areshown in Table 3. The highest amounts of gallic acid andferulic acid were found in Shi Ji Ba extracts, while the driedfruit extracts contained the lowest amounts of these com-pounds. In addition to phenolic acids, the other complexphenolic compounds in guava extracts may also providethe antioxidant acitiviy. However, further studies on theeffective components in guava extracts, which contributeto the antioxidant ability, are required.

Ferulic acid and its precursors, p-coumaric acid and caf-feic acid are synthesized in plants. Ferulic acid occurs incereals and vegetables, such as rice, wheat, oats, tomatoes,asparagus, olives and many other plants. Recently, a greatdeal of focus has been placed on the antioxidant potentiali-ties of ferulic acid and its n-alkyl esters (Anselmi et al., 2004;Kanski, Aksenova, Stoyanova, & Butterfield, 2002). San-chez-Moreno, Larrauri, and Saura-Calixto (1999) indicatedthat the inhibition of lipid oxidation of the phenolic com-pounds and antioxidant standards followed the order: rutin,ferulic acid > tannic acid, gallic acid, resveratrol > BHA,quercetin > tocopherol > caffeic acid, in a linoleic acid sys-tem. Meanwhile, the free radical-scavenging activity wasin the order: gallic acid > tannic acid, caffeic acid, quercetin,BHA, rutin > ferulic acid, tocopherol, resveratrol. In con-trast, several researches have indicated that ferulic acidwas ineffective, and even promoted the oxidation of low-density lipoprotein induced by copper (Chalas et al., 2001;Cirico & Omaye, 2006). In our results, gallic acid and ferulicacid may have important roles in the antioxidant activityand free radical scavenging ability of guava extracts.

3.4. Correlation analysis

Calculated coefficients of correlations between antioxi-dant activity, scavenging effects on radicals and contentsof phenolic compounds of guava extracts are shown inTable 4. The antioxidant activity of guava extracts was sig-

Table 4Correlation between the antioxidant properties and phenolic compounds of extracts from leaves and dried fruit of guava

Antioxidant activitya Scavenging effectsa Phenolic compounds

Superoxide anion ABTS�+ radicals Peroxyl radicals Total Gallic acid Ferulic acid

Antioxidant activity 1.000

Scavenging effectsSuperoxide anion 0.876* 1.000ABTS�+ radicals 0.880* 0.991* 1.000Peroxyl radicals 0.853** 0.921* 0.944* 1.000

Phenolic compoundsTotal 0.797** 0.927* 0.909* 0.875* 1.000Gallic acid 0.810** 0.759** 0.698 0.585 0.688 1.000Ferulic acid 0.685 0.763** 0.729 0.631 0.682 0.895* 1.000

a The concentrations of guava extracts in the antioxidant activity, scavenging effects on superoxide anion, ABTS�+ radicals and peroxyl radicals used forcorrelation analysis were 50, 200,10, 2.5 lg/ml, respectively.

* P < 0.01.** P < 0.05.

nificantly correlated with their scavenging effects on super-oxide anion (P < 0.01), ABTS�+ radicals (P < 0.01) andperoxyl radicals (P < 0.05). Therefore, the antioxidantactivities of guava extracts may be due to their scavengingeffects on radicals and blocking of the chain reaction in theperoxidation of linoleic acid. For scavenging effects on rad-icals, high correlations (R = 0.921–0.991) were observedbetween various radicals, indicating that these three meth-ods have satisfactory correlations for the examination ofantioxidants. The antioxidant activity (P < 0.05) and scav-enging effects on superoxide anion, ABTS�+ radicals andperoxyl radicals (P < 0.01) of guava extracts was also wellcorrelated with their contents of total phenolic compounds.The same trends were observed in the correlation of thecontent of gallic acid and the antioxidant activity and scav-enging effects on superoxide anion. According to recentreports, a highly positive relationship existed between totalphenolics and antioxidant activity in many plant species(Dasgupta & De, 2004; Dorman & Hiltunen, 2004).

4. Conclusions

In the present study, the extracts from guava were foundto possess strong antioxidant activity. The antioxidantmechanisms of guava leaf extracts may be attributed totheir free radical-scavenging ability. In addition, phenoliccompounds appear to be responsible for the antioxidantactivity of guava extracts. On the basis of the resultsobtained, guava extracts from either the leaf or dried fruitcan be used for a variety of beneficial chemo-preventiveeffects. However, further studies on the antioxidative com-ponents of guava extracts and more in vivo evidence fromdiabetic patients are required.

Acknowledgement

This work is part of a Research Project, NSC 88-2313-B-166-001, supported by the National Science Council,Republic of China.

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